Efficient generation of insect-based cell-free translation extracts active in glycosylation and signal sequence processing

A novel method for generation of insect-based cell-free translation extracts is presented. The protocol can be completed in less than an hour, and the resulting extracts are extremely proficient in N-linked glycosylation and signal sequence processing. No specialized equipment other than that usually present in an ordinary biochemistry laboratory is required. The novel approach dramatically reduces cost and time while rendering enhanced lysates compared to previously published strategies.     

New Mobilizable Vectors Suitable for Gene Replacement in Gram-Negative Bacteria and Their Use in Mapping of the 3′ End of the Xanthomonas campestris pv. campestris gum Operon

We describe useful vectors to select double-crossover events directly in site-directed marker exchange mutagenesis in gram-negative bacteria. These vectors contain the gusA marker gene, providing colorimetric screens to identify bacteria harboring those sequences. The applicability of these vectors was shown by mapping the 3′ end of the Xanthomonas campestris gum operon, involved in biosynthesis of xanthan.     

Endoscopic approach for the resection of forehead masses

Over the past several years, surgery aided by the endoscope has come into favor for a number of reasons. Because it is minimally invasive surgery, it has less morbidity, thus, reduced postoperative pain and complications. It results in earlier mobilization and shorter hospitalization, and most importantly, it contributes to an improved cosmetic appearance as a result of a shortened incision line concealed within the hairline in most cases. We have proposed an alternative approach to the surgical resection of forehead masses by means of the endoscope, which has proven to be useful not only for diagnosis but also as a therapeutic tool for the removal of forehead lesions. This report described the clinical experience with the removal of forehead masses in four patients. The cases illustrated the feasibility and ease of resecting a variety of forehead masses with excellent cosmetic results. We hope that more plastic surgeons will use the proposed technique and will continue to explore the safe limits of endoscopic plastic surgery.     

Xanthan gum biosynthesis and application: a biochemical /genetic perspective

Xanthan gum is a complex exopolysaccharide produced by the plant-pathogenic bacterium Xanthomonas campestris pv. campestris. It consists of D-glucosyl, D-mannosyl, and D-glucuronyl acid residues in a molar ratio of 2:2:1 and variable proportions of O-acetyl and pyruvyl residues. Because of its physical properties, it is widely used as a thickener or viscosifier in both food and non-food industries. Xanthan gum is also used as a stabilizer for a wide variety of suspensions, emulsions, and foams. This article outlines aspects of the biochemical assembly and genetic loci involved in its biosynthesis, including the synthesis of the sugar nucleotide substrates, the building and decoration of the pentasaccharide subunit, and the polymerization and secretion of the polymer. An overview of the applications and industrial production of xanthan is also covered. Received: 18 March 1998 / Received revision: 29 April 1998 / Accepted: 30 April 1998     

Infectious salmon anaemia virus (ISAV) isolated from the ISA disease outbreaks in Chile diverged from ISAV isolates from Norway around 1996 and was disseminated around 2005, based on surface glycoprotein gene sequences

Background

All viruses in the family Bunyaviridae possess a tripartite genome, consisting of a small, a medium, and a large RNA segment. Bunyaviruses therefore possess considerable evolutionary potential, attributable to both intramolecular changes and to genome segment reassortment. Hantaviruses (family Bunyaviridae, genus Hantavirus) are known to cause human hemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome. The primary reservoir host of Sin Nombre virus is the deer mouse (Peromyscus maniculatus), which is widely distributed in North America. We investigated the prevalence of intramolecular changes and of genomic reassortment among Sin Nombre viruses detected in deer mice in three western states.

Methods

Portions of the Sin Nombre virus small (S) and medium (M) RNA segments were amplified by RT-PCR from kidney, lung, liver and spleen of seropositive peromyscine rodents, principally deer mice, collected in Colorado, New Mexico and Montana from 1995 to 2007. Both a 142 nucleotide (nt) amplicon of the M segment, encoding a portion of the G2 transmembrane glycoprotein, and a 751 nt amplicon of the S segment, encoding part of the nucleocapsid protein, were cloned and sequenced from 19 deer mice and from one brush mouse (P. boylii), S RNA but not M RNA from one deer mouse, and M RNA but not S RNA from another deer mouse.

Results

Two of 20 viruses were found to be reassortants. Within virus sequences from different rodents, the average rate of synonymous substitutions among all pair-wise comparisons (π_s) was 0.378 in the M segment and 0.312 in the S segment sequences. The replacement substitution rate (π_a) was 7.0 × 10^-4 in the M segment and 17.3 × 10^-4 in the S segment sequences. The low π_a relative to π_s suggests strong purifying selection and this was confirmed by a Fu and Li analysis. The absolute rate of molecular evolution of the M segment was 6.76 × 10^-3 substitutions/site/year. The absolute age of the M segment tree was estimated to be 37 years. In the S segment the rate of molecular evolution was 1.93 × 10^-3 substitutions/site/year and the absolute age of the tree was 106 years. Assuming that mice were infected with a single Sin Nombre virus genotype, phylogenetic analyses revealed that 10% (2/20) of viruses were reassortants, similar to the 14% (6/43) found in a previous report.

Conclusion

Age estimates from both segments suggest that Sin Nombre virus has evolved within the past 37–106 years. The rates of evolutionary changes reported here suggest that Sin Nombre virus M and S segment reassortment occurs frequently in nature.